Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse

The cortical actin cytoskeleton has been shown to be critical for the reorganization and heterogeneity of plasma membrane components of many cells, including T cells. Building on previous studies at the T cell immunological synapse, we quantitatively assess the structure and dynamics of this meshwork using live-cell superresolution fluorescence microscopy and spatio-temporal image correlation spectroscopy. We show for the first time, to our knowledge, that not only does the dense actin cortex flow in a retrograde fashion toward the synapse center, but the plasma membrane itself shows similar behavior. Furthermore, using two-color, live-cell superresolution cross-correlation spectroscopy, we demonstrate that the two flows are correlated and, in addition, we show that coupling may extend to the outer leaflet of the plasma membrane by examining the flow of GPI-anchored proteins. Finally, we demonstrate that the actin flow is correlated with a third component, alpha-actinin, which upon CRISPR knockout led to reduced plasma membrane flow directionality despite increased actin flow velocity. We hypothesize that this apparent cytoskeletal-membrane coupling could provide a mechanism for driving the observed retrograde flow of signaling molecules such as the TCR, Lck, ZAP70, LAT, and SLP76.